TLR7/8 Agonists-based Multimodal Nano-delivery Platform For Tumor-specific Immunotherapy

Cancer is a serious threat to human health and the development of therapeutic strategies for cancer has become one of the key research areas in the treatment of the disease in China.The journal Science published cancer immunotherapy as one of the top ten technological breakthroughs of the year,revealing the enormous potential for clinical application of immunotherapy.Cancer immunotherapy is a treatment that restores the body’s anti-tumour immune response by restarting and maintaining the cancer-immunity cycle,thereby controlling and eliminating tumours.However,the results of clinical studies have shown that only a small proportion of patients currently benefit from this,and most patients are not responsive to immunotherapy strategies.The "cold" nature of tumors,characterized by inadequate infiltration and failed priming T lymphocytes,is a major cause of low response rates and the greatest barrier to the generalization of immunotherapy benefits.Therefore,turning the tumor from "cold" to "hot" via enhancing the intratumoral infiltration and activation of T lymphocytes will become an important breakthrough in the popularization of tumor therapy.Dendritic cells(DC),a professional antigen-presenting cells,are key mediators of immune responses and play an important role in the initiation and regulation of innate and adaptive immune responses.In the tumor microenvironment,DC can take up,process and present tumour antigens and provide co-stimulatory signals to activate T-cell immunity,thus manipulating DC has great potential to induce effective anti-tumor immunity.Toll-like receptor(TLR),an important class of pattern recognition receptors(PRRs)in innate immunity,can detect local infection and tissue damage,thereby preventing systemic infection and monitoring the production of malignant cells.Activation of TLR in DC cells can induce myeloid differentiation factor 88(MyD88)or domain-containing adaptor-inducing interferon-β(TRIF)dependent signaling pathways that activate nuclear factors κB(NF-κB).The secretion of cytokines and chemokines can activate innate immune responses and mediate acquired immune responses,remodeling the tumor microenvironment.Among the many TLR isoforms,agonists aimed at TLR3,TLR4,TLR7/8,and TLR9 are more widely studied in anti-tumor applications.Among these,TLR7/8 agonists can target all endogenous DC subpopulations to induce higher levels of pro-inflammatory factor secretion and upregulate co-stimulatory receptors.In addition,TLR7/8 agonists can activate both TLR7 and TLR8,demonstrating greater clinical translational potential.Imidazoquinolines are the most commonly reported TLR7/8 agonists,of which Imiquimod(IMQ)has been approved by the US FDA for basal cell carcinoma(topical application).Resiquimod(R848)is produced by adding hydroxyl and ethoxymethyl chains to IMQ and exhibits a more potent TLR8 agonist effect.Sunil A.David’s team developed a novel TLR7/8 agonist(1-(4-(aminomethyl)benzyl)-2-butyl-lH-imidazo[4,5-c]quinolin-4-amine,IMDQ)that is more efficient and easily modifiable based on conformational studies.However,immunotherapeutic strategies using TLR7/8 agonists as a single component still fail to elicit a potent T lymphocyte immune response.The combination of TLR7/8 agonist with other functional agents holds promise for enhancing DC function and achieving multimodal immunomodulation,thereby inducing an enhanced cellular immune response.In recent years,immunogenic cell death(ICD)effects triggered by certain chemotherapy drugs(such as Oxaliplatin(OXA),photothermal therapy(PTT),and chemodynamic therapy(CDT)have aroused widespread interest among researchers.It can promote the eversion of the "eat me" signal calreticulin(CRT),the secretion of the "find me" signal adenine triphosphate(ATP),and the release of the high mobility group box 1 protein(HMGB1),effectively enhancing the function of DC,thus enhancing the intratumoral infiltration of T lymphocytes.Therefore,the rational and safe application of ICD inducers to tumor treatment is of great significance in activating the body’s anti-tumor immunity and producing long-term anti-tumor effects.However,due to the non-specific distribution characteristics of the above chemotherapeutic drugs and immunomodulatory small molecules after injection,their in vivo application tends to induce systemic non-selective cytotoxicity and immune activation,which greatly limits the clinical application of these drugs.Thus,the precise delivery of drugs in specific tissues or cells is essential to ensure a safe and effective immune response..Nano-based drug delivery systems(NDDS)have potential clinical applications due to their improved pharmacokinetic properties,tumor-targeted delivery capabilities,and responsive drug release within the tumor microenvironment or tumor cells,which can achieve better anti-tumor effects.At present,many NDDS-based drug formulations have been approved in clinic and have significantly improved the effectiveness of oncology treatment.Among them,polymer-based nanocarriers are generally composed of amphiphilic block copolymers with both hydrophilic and hydrophobic ends,having good self-assembly capability,well-defined structure,and high drug loading capacity.It mainly includs polymer micelles,polymer vesicles,and polymer-drug conjugates,in which the polymer-drug conjugates show great potential for drug delivery due to their ease of modification,controlled drug delivery and stable properties.The reversible addition fracture chain transfer(RAFT)polymerization technology,a widely reported polymerization tolls,provides a universal platform for the on-demand design of polymer-drug conjugates.Based on the above discussion,the following research has been carried out on this topic:Part Ⅰ:Tumor-selective activation of TLR7/8 agonist nanoimmunomodulatory systems for tumour-specific immune activation.In this section,to address the issue of systemic immune activation of small molecule TLR7/8 agonist upon systemic injection,we synthesized the tumor-responsive drug monomer IMQ-HEDSMA using chemical means to temporarily mask its key active site(amino group at C4).Then,the monomer was polymerized via RAFT polymerization to obtain block copolymer pDMA-b-pIMQ.To relieve the immunosuppressed state of T lymphocytes,a block copolymer of pDMA-b-pJQl with immune checkpoint inhibitor JQ1 conjugate was constructed by the same method.To further achieve its tumor targeting ability,we modified a tumor targeting peptide c-RGD on the polymer end group,and obtained c-RGD-pDMA-b-pIMQ and c-RGD-pDMA-b-pJQl.The above tumour-targeting block polymers can spontaneously form nanoscale pre-drug immune modulators c-N@IM/JQ.Upon intravenous injection,c-N@IM/JQ accumulated at the tumor site with the guild of c-RGD peptide.The rich glutathione(GSH)within tumors promoted the release of IMQ and JQ1.Released IMQ can effectively promote the maturation of DC,thereby promoting intratumoral infiltration of T lymphocytes;JQ1 can further relieve the immunosuppressive signals of T lymphocytes,and both of them synergistically trigger a tumor specific immune response.1H-NMR spectroscopy demonstrated the synthesis and targeted modification of functional monomers and block copolymers;The particle size,zeta potential,TEM image,and release curve proved the successful construction of c-N@IM/JQ,and the tumor-triggered drug release;Cytotoxicity,cellular uptake,and BMDC activation demonstrated the immunomodulatory function of c-N@IM/JQ;IVIS imaging and flow cytometry demonstrated the tumor-targeted distribution of c-N@IM/JQ;In vivo tumor suppression experiments,immune infiltration analysis,and the pathological observations confirmed the anti-tumour effect and immune activation ability of c-N@IM/JQ.Part Ⅱ:A three-in-one multimodal nano-delivery platform of"Photothermal/chemotherapy/immunotherapy" for tumor-specific immunotherapy.Based on the immune effects of TLR7/8 agonists,in this section,we further combined with the ICD effects induced by PTT and OXA to construct a novel multimodal nano-delivery platform of "photothermal/chemotherapy/immunotherapy".We selected hollow copper sulfide(CuS)nanoparticles as photothermal therapeutic agents and loaded with chemotherapeutic agent OXA to form OXA@CuS,both of which have ICD effects and can be used as an amplifying combination for ICD.Then,a DC-targeted,TLR7/8 agonist-conjugated polymer-drug conjugates Man-pIMDQ was synthesized using RAFT polymerization.To prolong the tumor retention of nanoparticles,we synthesized tumor-targeted polymer FA-pDMA in the same way.Finally,we anchored FA-pDMA and Man-pIMDQ onto the surface of OXA@CuS to construct F/IMO@CuS.Upon intratumoral injection,F/IMO@CuS prolonged the intra-tumour retention time via the binding of FA with tumor cells.Under 808 nm laser irradiation,F/IMO@CuS gradually disintegrate while exerting photothermal therapy.The released OXA and PTT trigger the ICD effect of tumor together.ICD-triggered immune signaling and Man-pIMDQ synergistically promote the maturation of DC.Activated DC promotes the effective intratumoral infiltration of T lymphocytes to inhibit tumor progression,and meanwhile the immune memory is formed in vivo to prevent tumor recurrence.1H-NMR spectroscopy demonstrated the synthesis and targeted modification of functional monomers,polymers and polymer-drug conjugates;The particle size,zeta potential,and TEM images proved the successful construction of F/IMO@CuS;Cytotoxicity,cellular uptake,photothermal imaging,ICD signal detection,and BMDC activation demonstrated the photothermal,chemotherapy effect,ICD induction,and immunomodulatory function of F/IMO@CuS;IVIS imaging and flow cytometry demonstrated the enhanced intratumoral retention and intracellular distribution of F/IMO@CuS;In vivo tumor suppression experiments,immune infiltration analysis,and pathological observations confirmed the anti-tumour effect and immune activation ability of F/IMO@CuS.